The successful sequencing of the human genome has given rise to the next scientific opportunity of the twenty-first century: functional annotation of the proteome. About one-fifth of the genome encodes secretory proteins, a small number of which represent signaling molecules for G-protein coupled receptors (GPCRs). The major challenge that we will explore in this proposal is the comprehensive 'de-orphanization' (and thereby annotation) of the universe of peptides involved in neuronal GPCR signaling. There are currently about 100 non-olfactory orphan GPCRs, many of which are expected to use peptide ligands; however, fewer than a dozen novel peptides have been identified within the last eight years- and none at all within the last few years. Bioinformatics analyses indicate that the genome contains about 150 untested secretory proteins which possess biochemical similarities to known peptide precursors. We postulate that these proteins contain the missing peptide ligands for orphan GPCRs. However, in order to make these new orphan receptor- peptide matches, fresh approaches to peptide ligand identification are urgently needed. To identify novel peptide neurotransmitters we propose to take an innovative approach integrating expertise in bioactive peptide synthesis (Lindberg laboratory) with expertise in GPCR screening (Roth laboratory). We will experimentally confirm the presence of prohormone convertase-cleavable sites in a bioinformatically-derived list of putative precursors. Bioactive peptides will be generated from all validated precursors through large-scale in vitro posttranslational modification reactions using physiological enzymes (Lindberg laboratory). We will then discover cognate receptors to these peptides via functional screening against the entire genomic complement of known and orphan peptide receptors using facile screening technologies (Roth laboratory). Our results will enable us to match orphan receptors with novel peptide ligands, thus providing new neuropeptide-receptor signaling pairs. Since neuropeptide signaling pathways are critical to brain function and include pathways involved in mood and cognition, mental disorders, and drug reward, our results will significantly advance our understanding of mental health and disease, and may also generate new drug targets. While we will focus on obtaining and testing neuronally-expressed precursors/ligands and receptors, our research is also likely to uncover other ligand-receptor matches; thus a major impact on the many other physiological processes controlled by peptide- GPCR receptor signaling pathways is also anticipated.